Anti-Viral and Anti-Inflammatory Therapeutic Effect of RAGE-Ig Protein Against Multiple SARS-CoV-2 Variants of Concern Demonstrated in K18-hACE2 Mouse and Syrian Golden Hamster Models (preprint)

Significance: SARS-CoV-2 Variants of Concern (VOCs) continue to evolve and re-emerge with chronic inflammatory long-COVID sequelae necessitating the development of anti-inflammatory therapeutic molecules. Therapeutic effects of the Receptor for Advanced Glycation End products (RAGE) were reported in many inflammatory diseases. However, a therapeutic effect of the RAGE in COVID-19 has not been reported. In the present study, we investigated whether and how the RAGE-Ig fusion protein would have an anti-viral and anti-inflammatory therapeutic effect in the COVID-19 system. Methods: The protective therapeutic effect of RAGE-Ig was determined in vitro in K18-hACE2 transgenic mice and Syrian golden hamsters infected with six various VOCs of SARS-CoV-2. The underlying anti-viral mechanism of RAGE-Ig was determined in vitro in SARS-CoV-2-infected human lung epithelial cells (BEAS-2B). Results: Following treatment of K18-hACE2 mice and hamsters infected with various SARS-CoV-2 VOCs with RAGE-Ig, we demonstrated: (i) significant dose-dependent protection (i.e. greater survival, less weight loss, lower virus replication in the lungs); (ii) a reduction of inflammatory macrophages (F4/80+/Ly6C+) and neutrophils (CD11b+/Ly6G+) infiltrating the infected lungs; (iii) a RAGE-Ig dose-dependent increase in the expression of type I interferons (IFN-alpha;, and IFN-beta) and type III interferon (IFN lambda2) and a decrease in the inflammatory cytokines (IL-6 and IL-8) in SARS-CoV-2-infected human lung epithelial cells; and (iv) a dose-dependent decrease in the expression of CD64 (FcgR1) on monocytes and lung epithelial cells from symptomatic COVID-19 patients. Conclusion: Our pre-clinical findings revealed type I and III interferons-mediated anti-viral and anti-inflammatory therapeutic effects of RAGE-Ig protein against COVID-19 caused by multiple SARS-CoV-2 VOCs.

Cross-Protection Induced by Highly Conserved Human B, CD4+, and CD8+ T Cell Epitopes-Based Coronavirus Vaccine Against Severe Infection, Disease, and Death Caused by Multiple SARS-CoV-2 Variants of Concern (preprint)

Background: The Coronavirus disease 2019 (COVID-19) pandemic has created one of the largest global health crises in almost a century. Although the current rate of SARS-CoV-2 infections has decreased significantly; the long-term outlook of COVID-19 remains a serious cause of high death worldwide; with the mortality rate still surpassing even the worst mortality rates recorded for the influenza viruses. The continuous emergence of SARS-CoV-2 variants of concern (VOCs), including multiple heavily mutated Omicron sub-variants, have prolonged the COVID-19 pandemic and outlines the urgent need for a next-generation vaccine that will protect from multiple SARS-CoV-2 VOCs. Methods: In the present study, we designed a multi-epitope-based Coronavirus vaccine that incorporated B, CD4+, and CD8+ T cell epitopes conserved among all known SARS-CoV-2 VOCs and selectively recognized by CD8+ and CD4+ T-cells from asymptomatic COVID-19 patients irrespective of VOC infection. The safety, immunogenicity, and cross-protective immunity of this pan-Coronavirus vaccine were studied against six VOCs using an innovative triple transgenic h-ACE-2-HLA-A2/DR mouse model. Results: The Pan-Coronavirus vaccine: (i) is safe; (ii) induces high frequencies of lung-resident functional CD8+ and CD4+ TEM and TRM cells; and (iii) provides robust protection against virus replication and COVID-19-related lung pathology and death caused by six SARS-CoV-2 VOCs: Alpha (B.1.1.7), Beta (B.1.351), Gamma or P1 (B.1.1.28.1), Delta (lineage B.1.617.2) and Omicron (B.1.1.529). Conclusions: A multi-epitope pan-Coronavirus vaccine bearing conserved human B and T cell epitopes from structural and non-structural SARS-CoV-2 antigens induced cross-protective immunity that cleared the virus, and reduced COVID-19-related lung pathology and death caused by multiple SARS-CoV-2 VOCs.

High Frequencies of PD-1+TIM3+TIGIT+CTLA4+ Functionally Exhausted SARS-CoV-2-Specific CD4+ and CD8+ T Cells Associated with Severe Disease in Critically ill COVID-19 Patients (preprint)

SARS-CoV-2-specific memory T cells that cross-react with common cold coronaviruses (CCCs) are present in both healthy donors and COVID-19 patients. However, whether these cross-reactive T cells play a role in COVID-19 pathogenesis versus protection remain to be fully elucidated. In this study, we characterized cross-reactive SARS-CoV-2-specific CD4+ and CD8+ T cells, targeting genome-wide conserved epitopes in a cohort of 147 non-vaccinated COVID-19 patients, divided into six groups based on the degrees of disease severity. We compared the frequency, phenotype, and function of these SARS-CoV-2-specific CD4+ and CD8+ T cells between severely ill and asymptomatic COVID-19 patients and correlated this with alpha-CCCs and beta-CCCs co-infection status. Compared with asymptomatic COVID-19 patients, the severely ill COVID-19 patients and patients with fatal outcomes: (i) Presented a broad leukocytosis and a broad CD4+ and CD8+ T cell lymphopenia; (ii) Developed low frequencies of functional IFN-gamma-producing CD134+CD138+CD4+ and CD134+CD138+CD8+ T cells directed toward conserved epitopes from structural, non-structural and regulatory SARS-CoV-2 proteins; (iii) Displayed high frequencies of SARS-CoV-2-specific functionally exhausted PD-1+TIM3+TIGIT+CTLA4+CD4+ and PD-1+TIM3+TIGIT+CTLA4+CD8+ T cells; and (iv) Displayed similar frequencies of co-infections with beta-CCCs strains but significantly fewer co-infections with alpha-CCCs strains. Interestingly, the cross-reactive SARS-CoV-2 epitopes that recalled the strongest CD4+ and CD8+ T cell responses in unexposed healthy donors (HD) were the most strongly associated with better disease outcome seen in asymptomatic COVID-19 patients. Our results demonstrate that, the critically ill COVID-19 patients displayed fewer co-infection with alpha-CCCs strain, presented broad T cell lymphopenia and higher frequencies of cross reactive exhausted SARS-CoV-2-specific CD4+ and CD8+ T cells. In contrast, the asymptomatic COVID-19 patients, appeared to present more co-infections with alpha-CCCs strains, associated with higher frequencies of functional cross-reactive SARS-CoV-2-specific CD4+ and CD8+ T cells. These findings support the development of broadly protective, T-cell-based, multi-antigen universal pan-Coronavirus vaccines.

Deep immune profiling of the maternal-fetal interface with mild SARS-CoV-2 infection (preprint)

Pregnant women are an at-risk group for severe COVID-19, though the majority experience mild/asymptomatic disease. Although severe COVID-19 has been shown to be associated with immune activation at the maternal-fetal interface even in the absence of active viral replication, the immune response to asymptomatic/mild COVID-19 remains unknown. Here, we assessed immunological adaptations in both blood and term decidua from 9 SARS-exposed pregnant women with asymptomatic/mild disease and 15 pregnant SARS-naive women. In addition to selective loss of tissue-resident decidual macrophages, we report attenuation of antigen presentation and type I IFN signaling but upregulation of inflammatory cytokines and chemokines in blood monocyte derived decidual macrophages. On the other hand, infection was associated with remodeling of the T cell compartment with increased frequencies of activated CD69+ tissue-resident T cells and decreased abundance of Tregs. Interestingly, frequencies of cytotoxic CD4 and CD8 T cells increased only in the blood, while CD8 effector memory T cells were expanded in the decidua. In contrast to decidual macrophages, signatures of type I IFN signaling were increased in decidual T cells. Finally, T cell receptor diversity was significantly reduced with infection in both compartments, albeit to a much greater extent in the blood. The resulting aberrant immune activation in the placenta, even with asymptomatic disease may alter the exquisitely sensitive developing fetal immune system, leading to long-term adverse outcomes for offspring.

Antibody Response to a Nucleocapsid Epitope as a Marker for COVID-19 Disease Severity (preprint)

Characterization of antibody response to SARS-CoV-2 is urgently needed to predict COVID-19 disease trajectories. Ineffective antibodies or antibody-dependent enhancement (ADE) could derail patient immune responses, for example. ELISA and coronavirus antigen microarray (COVAM) analysis epitope-mapped plasma from 86 COVID-19 patients. The experiments identified antibodies to a 21-residue epitope from nucleocapsid (termed Ep9) associated with severe disease, including ICU stay, requirement for ventilators, and death. Furthermore, anti-Ep9 antibodies correlate both with various comorbidities and ADE hallmarks, including increased IL-6 levels and early IgG response. Importantly, anti-Ep9 antibodies can be detected within five days post-symptom onset and sometimes within one day. The results lay the groundwork for a new type of COVID-19 diagnostic for the early prediction of disease severity to guide more effective therapeutic interventions.

Genome-Wide Asymptomatic B-Cell, CD4+ and CD8+ T-Cell Epitopes, that are Highly Conserved Between Human and Animal Coronaviruses, Identified from SARS-CoV-2 as Immune Targets for Pre-Emptive Pan-Coronavirus Vaccines (preprint)

Over the last two decades, there have been three deadly human outbreaks of Coronaviruses (CoVs) caused by emerging zoonotic CoVs: SARS-CoV, MERS-CoV, and the latest highly transmissible and deadly SARS-CoV-2, which has caused the current COVID-19 global pandemic. All three deadly CoVs originated from bats, the natural hosts, and transmitted to humans via various intermediate animal reservoirs. Because there is currently no universal pan-Coronavirus vaccine available, two worst-case scenarios remain highly possible: (1) SARS-CoV-2 mutates and transforms into a seasonal "flu-like" global pandemic; and/or (2) Other global COVID-like pandemics will emerge in the coming years, caused by yet another spillover of an unknown zoonotic bat-derived SARS-like Coronavirus (SL-CoV) into an unvaccinated human population. Determining the antigen and epitope landscapes that are conserved among human and animal Coronaviruses as well as the repertoire, phenotype and function of B cells and CD4+ and CD8+ T cells that correlate with resistance seen in asymptomatic COVID-19 patients should inform in the development of pan-Coronavirus vaccines 1. In the present study, using several immuno-informatics and sequence alignment approaches, we identified several human B-cell, CD4+ and CD8+ T cell epitopes that are highly conserved in: (i) greater than 81,000 SARS-CoV-2 human strains identified to date in 190 countries on six continents; (ii) six circulating CoVs that caused previous human outbreaks of the "Common Cold"; (iii) five SL-CoVs isolated from bats; (iv) five SL-CoV isolated from pangolins; (v) three SL-CoVs isolated from Civet Cats; and (vi) four MERS strains isolated from camels. Furthermore, we identified cross-reactive asymptomatic epitopes that: (i) recalled B cell, CD4+ and CD8+ T cell responses from both asymptomatic COVID-19 patients and healthy individuals who were never exposed to SARS-CoV-2; and (ii) induced strong B cell and T cell responses in "humanized" Human Leukocyte Antigen (HLA)-DR/HLA-A*02:01 double transgenic mice. The findings herein pave the way to develop a pre-emptive multi-epitope pan-Coronavirus vaccine to protect against past, current, and potential future outbreaks.